FGFR2b is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation

Summary Exocrine secretory acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific epithelial SG populations later in development and during acinar differentiation are unknown. Here, we predicted FGFR dependence in specific populations using scRNAseq data and conditional mouse models to delete FGFRs in vivo. We identifed essential roles for FGFRs in craniofacial and early SG development, as well as progenitor function during duct homeostasis. Importantly, we discovered that FGFR2b was critical for seromucous and serous acinar cell differentiation and secretory gene expression (Bpifa2 and Lpo) via MAPK signaling, while FGFR1b was dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activated the FGFR2b-dependent seromucous transcriptional program. We propose a model where MEC-derived FGF7 drives seromucous acinar differentiaton, providing a rationale for targeting FGFR2b signaling in regenerative therapies to restore acinar function.


Introduction
Exocrine glands secrete fluids essential for maintenance of their target tissues 1 . Exocrine SGs are critical for oral health as they generate saliva for mastication, maintenance of the oral microbiome, and lubrication of the oral cavity. There are three major mammalian SGs, submandibular (SMG), sublingual (SLG), parotid (PG) as well as minor salivary glands (MSG) which all differentiate in the composition of saliva they secrete 2,3 . The bulk of saliva secretion comes from highly specialized acinar cells defined by the secretory proteins they produce and are either seroumucous, mucous or serous depending on the protein contents of the saliva. Despite the central role of acinar cells, not much is known about the developmental mechanisms that drive acinar cells to either produce a mucous containing saliva compared with watery serous saliva. In vivo, acinar cells are surrounded by contractile myoepithelial cells (MEC) that wrap around and directly contact the acini, and connected duct cells that modify and transport saliva into the oral cavity. Loss of acinar cells is a common feature of pathologies including autoimmune diseases and as a side effect of irradiation therapy, and acinar regeneration continues to be a major clinical challenge. Preclinical studies have shown through lineage tracing that acinar and MECs are selfmaintained, while basal duct cells are restricted progenitors during homeostasis 4 . Following injury, all cell compartments have regenerative potential, however, the contribution from any lineage is dependent on the degree and type of injury 4 . This has resulted in an updated view of stemness and plasticity in SGs leading to a renewed focus on niche signals and microenvironments that may inform regenerative therapies 5 .
Fibroblast growth factor receptors (FGFR) are a family of four receptor tyrosine kinases involved in development, progenitor cell proliferation, and tumorigenesis of multiple organ systems including SGs 6,7 . The critical role for FGFR signaling in human SGs is evident by haploinsufficiency of either FGFR2b or one of its major ligands, FGF10, which leads to two rare genetic diseases, aplasia of lacrimal and salivary glands (ALSG: MIM #180929) and lacrimoauriculo-dento-digital syndrome (LADD: MIM #149730) 8 . Studies using murine models have established that both FGFR1b and FGFR2b are expressed in the epithelium during embryonic development and the ligands, FGF10 and FGF7, are expressed in the mesenchyme. Paracrine signaling between FGF10 in the mesenchyme and FGFR2b in the epithelium is required and sufficient for SG initiation, while FGF7 is dispensable for gland initiation [9][10][11] . Reduction in FGFR1b signaling leads to decreased branching morphogenesis and smaller glands 12 . Hypoplastic glands are found in Fgf10 +/and Fgfr2b +/mice 13,14 , while ligand-dependent-gain-of-function, due to an Fgfr2b mutation (Fgfr2 +/Neo-S252W ) leads to hyperplasia 15,16 , suggesting sensitivity to FGF and FGFR protein expression levels. Although SGs, like many other branched organs, are dependent on FGFR signaling for early development, the role of FGFRs in cell-specific lineages, progenitor function and differentiation of specific acinar cell types are not known.
To investigate the role of FGFR signaling in specific SG cell types, we leveraged existing single-cell RNA-sequencing (scRNAseq) datasets [17][18][19][20] and confirm that human and mouse SG have similar expression patterns of both FGFR and FGF ligands. We used mice carrying floxed FGFR alleles with epithelial Cre drivers to conditionally delete FGFRs in specific cell populations in vivo. We confirmed the essential role of FGFR2b in the primary endbud for gland initiation and identified requirement of FGFRs in adult duct progenitor function. We discovered that FGFR2b signaling drives seromucous and serous acinar differentiation in the SMG and SLG, respectively, while FGFR1b is dispensable. Further investigation using ex vivo organ culture and loss and gain of function approaches identified the FGFR signaling was via MAPK signaling and the seromucous acinar transcriptional program was stimulated by FGF7, which is produced by adult MECs.
We focused our analysis on Fgfr1b and Fgfr2b due to their higher expression in both embryonic and postnatal development, compared to other Fgfr genes. In situ hybridization in postnatal glands confirmed the enrichment in Krt5+ cells, which include basal ducts and MECs ( Figure 1C, Figure S1B). There was also widespread Fgfr1b and Fgfr2b coexpression with Bhlha15+ (MIST1), a canonical acinar cell marker ( Figure 1D). Notably, at P1, acinar cells were enriched for either Fgfr1b or Fgfr2b, while adult acinar cells were positive for both receptors ( Figure 1D).
Taken together, these data show that Fgfr1b and Fgfr2b are the most widely expressed FGFRs in SG epithelium. They are enriched in MECs, basal duct and acinar cells, all populations which have progenitor potential during regeneration and have been shown to self-renew during adult SG homeostasis. Based on these findings, we predicted that both FGFR1B and FGFR2B signaling are important for SG epithelial cell development and critical in specific lineages and cell populations at later stages of development. We sought to test this hypothesis using bioinformatic data to direct studies with genetic mouse models and explant culture. Based on previous global FGFR knockout studies showing differential roles for FGFR1b and FGFR2b at different stages of development, we predicted that deleting Fgfr1b in the entire E12 epithelium would not affect gland initiation, while Fgfr2b would be required.

Genetic deletion of
Endbud and duct cells in the E12 epithelium can be identified bioinformatically by enrichment of Sox10 and Krt5, respectively (Figure 2A, B). At this stage, both populations expressed Fgfr1b and Fgfr2b along with the ectodermal transcription factor AP-2 (Tfap2a or Crect) and the epithelial marker Krt14 ( Figure 2B). Based on these data, we used the ectodermspecific Tfap2a-Cre (Crect) or the epithelial specific Krt14Cre mouse strains to delete Fgfr1b and Fgfr2b in vivo. Each of these strains were crossed with mice carrying floxed alleles for Fgfr1b and Fgfr2b in addition to the cell membrane-targeted, two-color fluorescent Cre-reporter mTmG mouse strain ( Figure 2C). This generates embryos where Cre+ cells have Fgfr deletions and express cell membrane-localized GFP. Thus, for further analysis, GFP was used as a pseudomarker for Fgfr manipulation.
Both Cre models generated embryos with gross morphological changes that were nonviable due to lack of organ development upon  SGs from the E12 Crect+;Fgfr1 fl/fl ;Fgfr2 fl/+ were comparable to control and formed a SMG with a stratified, invaginating epithelium in the condensing mesenchyme ( Figure 2E). In embryos with loss of two alleles of Fgfr2b, initiation of both the SMG and SLG occurred, appearing as an infolding of the epithelium, but the primary endbud failed to enlarge. The fold in the oral epithelium appeared similar to an early E11 SMG, but failed to form a stratified endbud ( Figure   2E). Similarly, Krt14Cre+;Fgfr1 fl/fl ;Fgfr2 fl/+ glands were comparable to control while Krt14Cre+;Fgfr1 fl/+ ;Fgfr2 fl/fl glands had a thickening of the oral epithelium, but had not stratified to form an enlarged endbud further shown by an almost complete absence of Sox10+ endbud cells ( Figure 2F). Mesenchymal SOX10+ expression in neural crest cells, precursors to the parasympathetic ganglion, was not affected ( Figure 2F). For both Crect and Krt14-Cre strains, no salivary gland was observed at E16, indicating that there was not simply a delay in development, but an absence of SG formation (data not shown).
Taken together, these data confirm the central role of Fgfr2b in gland initiation and demonstrate that Fgfr1b is dispensable for endbud formation. Further, to investigate cell specific roles in lineage specification, FGFRs must be deleted in duct and acinar cells directly using additional cre models.  Previous work has shown that this cross leads to viable mice; however, they develop a progressive skin phenotype 22 . Indeed, SGs were present in adult mice although they were 50% smaller in  To further analyze FGFR expression during acinar differentiation we bioinformatically isolated and re-clustered acinar cells from E16 and P1, resulting in 4 distinct clusters ( Figure 4A and Figure  In general, all markers had higher expression level at P1 compared to E16. Smgc, Bpifa2 and Lpo were enriched in their respective populations at both E16 and P1; however, some markers were stage-specific, such as Tesc at E16 and Gstt1 at P1 in Fgfr1b+ cells and Mucl2, Car6 and Prol1 in Fgfr2b+ cells at P1 ( Figure S3B).
The onset of acinar specification and the relative expression of markers for the specific populations were confirmed at three timepoints (E14, E15 and E16) using qPCR ( Figure 4D).
Immunostaining for canonical acinar specification markers showed detection at E15 with a progressively increasing organization of luminal AQP5, basolateral CLAUDIN10 (CLDN10) and nuclear MIST1 ( Figure 4E). Markers for the two subpopulations could be detected from E16, and by late E16 both populations were clearly distinguished with immunostaining for SMGC and LPO proteins ( Figure 4F). Furthermore, in P1 glands, the two pro-acinar populations could be visualized through immunostaining with either SMGC and LPO or GSTT1 and MUC10 ( Figure 4G, H). Costaining of cells with SMGC and GSTT1 or co-staining with LPO and MUC10 further confirmed they were expressed in the same cell populations ( Figure S3C, D). Based on average expression, cluster 2 ( Figure 4C) appeared to be double positive for Fgfr1b and Fgfr2b; however, this cluster contained cells either Smgc+ or Bpifa2+, and was defined by proliferative markers such as Mki67, Bub1b, Aurka and Top2a ( Figure S3E). Pathway analysis indicated active proliferation as the major functional state of these cells ( Figure S3E). Furthermore, proliferating acinar cells were found within both subpopulations (clusters 0 and 1, Figure 4I), indicating that cluster 2 is mainly proliferating cells made up by a mix of the two distinct populations rather than a Fgfr1b and Fgfr2b double positive population ( Figure 4I). Taken together, Fgfr1b and Fgfr2b are enriched within specific acinar subpopulations, and we hypothesized that FGFR signaling would differentially affect expression of defining markers and either the development or maturation of these acinar subpopulations.

Fgfr2b is required for seromucous and serous acinar differentiation through MAPK pathway
To test the direct effect of FGFR signaling on acinar differentiation, we used the Aqp5- staining in mucous cells was evident in all genotypes ( Figure 5I). Taken together, this data shows that Fgfr1b is dispensable while Fgfr2b is required for differentiation and expression of secretory markers in seromucous and serous cells in the SMG and SLG, respectively. It also highlights that mucous acinar cell differentiation is independent of FGFR signaling.
We utilized WT mice (ICR) and an organ culture system to further manipulate downstream signaling required for FGFR-dependent seromucous acinar differentiation. Isolated E15 SMGs from ICR mice were cultured for 24 and 48 hrs to establish the baseline gene expression during culture conditions ( Figure S5A). This showed consistent expression of Fgfr1b, Fgfr2b, Fgf10 and Aqp5 expression was also reduced after MAPK inhibition, indicating a specific MAPK signaling dependence for Aqp5 ( Figure 6C). As expected, Smgc, Tesc and Lman1 expression were not negatively affected by inhibitor treatments ( Figure 6C). Bpifa2 was decreased after FGFR, MAPK and PI3K inhibitor treatment, while Lpo showed a similar trend, while PLCγ inhibitor treatment did not affect Fgfr2b-dependent genes ( Figure 6C). Gross histology of all groups was comparable to control ( Figure S5G). After 24hrs treatment, luminal AQP5, nuclear MIST1 and lateral CLDN10, were detected in all groups indicating that early acinar differentiation was not lost ( Figure 6D). Staining for SMGC showed expression of the protein in all groups, while LPO was not detected after FGFR or MAPK inhibitor treatment ( Figure 6E). These results show that differentiation of seromucous acinar cells requires FGFR2b signaling through the MAPK pathway.

Seromucous acinar transcriptional program can be activated by FGF7, which is expressed by MEC that contact acini, as well as FGF10 produced by fibroblasts
Both FGF7 and FGF10 are major ligands for FGFR2b signaling 26,27 and activation of the acinar transcriptional program has therapeutic potential for regenerating exocrine secretory cells following injury or disease. Accordingly, we asked whether FGF7 and FGF10 could increase expression of Fgfr2b-dependent secretory markers. Initial experiments using E15 organ culture treated with additional exogenous FGF7 or FGF10 showed no increases in acinar gene expression compared to vehicle after 6 hrs, likely due to the robust endogenous FGF production (data not shown). Therefore, we performed a gain-of-function experiment to test whether the reduced seromucous differentiation after MAPK inhibition could be restored or increased by addition of exogenous FGF7 or FGF10. E15 glands were treated with MAPK inhibitor for 24 hrs before changing to fresh media with either MAPK inhibitor, the ligands FGF7 or FGF10 or a DMSO vehicle control ( Figure 7A). Treatment with MAPK inhibitor for 48 hrs decreased Aqp5, Bpifa2 and Lpo and washout with media containing vehicle control increased and partially rescued expression of all three genes ( Figure 7B). However, addition of FGF7 further increased the expression of both Aqp5 and Lpo above control expression, while Bpifa2 expression was restored to control levels ( Figure 7B). Addition of FGF10 did not increase Aqp5 but did increase Lpo expression ( Figure 7B). Inhibitor treatment for 48 hrs decreased expression of Smgc and addition of ligands showed an increasing trend although not significant compared to washout alone ( Figure   7B). Furthermore, immunostaining confirmed an increase of LPO staining following stimulation with either FGF7 or FGF10 after MAPK inhibition. In contrast, SMGC immunostaining decreased, and washout reversed this although ligand treatment did not result in further increases compared with washout alone (Figure 7C, D). These results show that MAPK-dependent seromucous differentiation (LPO expression and protein staining) can be stimulated ex vivo by both FGF7 and FGF10.
Since activation of FGFR2b is critical for seromucous differentiation, we asked which cells are the in vivo source of these ligands. The acinar niche, or microenvironment contains multiple cells producing the signals needed for homeostatic acinar function. Signals from MECs, nerves, blood vessels, immune cells, and fibroblasts all contribute to the acinar niche. Both Fgf7 and Fgf10 were detected by scRNAseq in cells within the acinar niche. As expected, fibroblasts expressed both Fgf7 and Fgf10, however surprisingly, Fgf7 was also predominantly expressed in MECs, while Fgf10 is expressed in mature ductal ionocytes and fibroblasts ( Figure 7E), as recently reported 28 . In vivo, MECs directly contact and wrap around the acini, and this acinar complex is surrounded by the basement membrane, whereas, the stromal fibroblasts that produce FGF10 are separated from acini by the basement membrane. Interestingly, human SGs showed a similar expression pattern, with FGF10 in fibroblasts and FGF7 expressed in MECs of SMG and MSG, but not detected by scRNAseq in PG MECs ( Figure 7F). We confirmed the novel expression of Fgf7 in MECs and fibroblasts in both P1 and adult SMGs through in situ hybridization ( Figure   7G). This suggests that FGF7 from MECs, in direct contact to acini, may provide critical niche signals for seromucous acinar differentiation. Taken together, we have shown that differentiation of seromucous acinar cells is dependent on FGFR2b-MAPK signaling via FGF7 and FGF10 from MECs as well as fibroblasts ( Figure 7H).

Discussion
We have previously defined roles for FGFR signaling during fetal ex vivo SMG branching morphogenesis in organ explant culture 29 . Here, we used genetic tools to conditionally delete The proposed mechanism in these reports include protecting acinar cells from apoptosis and stimulating the basal duct progenitor pool to differentiate into secretory acinar cells. The protective effects of FGF7 were proposed to include maintaining acinar, MEC and endothelial markers as well as saliva flow 47,48 . Taken together, these reports suggest an important role for FGF7-FGFR2b signaling in several cell types after irradiation damage and highlights potential for increasing acinar differentiation during tissue regenerative strategies.
Our findings provide genetic in vivo evidence that FGFR2b signaling will be required for stem cell-based or organoid-based regenerative therapies that will require seromucous acinar and serous differentiation as part of fully functional SG regeneration. It is likely that both or sequential EGFR and FGFR inputs will be required to drive acinar specification and subsequent seromucous acinar cell differentiation. Thus, we propose an central role for FGF7-FGFR2b signaling allowing for differentiation and maturation of specific acinar cell types, acinus morphology, as well as duct progenitor function.

Limitations of the study
Whether there are differential roles of FGFR1b and FGFR2b in basal duct cells remains to be determined. Although implied, acinar cell secretory volume was not directly measured in mice since ACID-Cre+;Fgfr2 fl/fl animals did not survive more than three days after birth. Our data highlight that mucous acinar differentiation in the SLG was not FGFR-dependent, and the signaling pathways that drive mucous secretory cell differentiation remain to be determined. The effect of FGFR signaling on MEC development and function was not directly addressed in the models used here, although recent evidence shows that MEC differentiation is driven by neurotrophin signaling. Lastly, this study does not address the FGFR-dependence of cellular plasticity following injury.

Acknowledgments
The authors would like to thank Elsa Berenstein, NIDCR imaging core (ZIC DE000750) and NIDCR veterinary resource core (ZIC DE000740) for excellent technical assistance. We thank

Declaration of interests
The authors declare no competing interests.

Lead Contact
Further information and requests for resources should be directed to Marit H. Aure (marit.aure@nih.gov).

Materials availability
No specific materials were generated in this work, see Supplemental Table 1 for resources.

Data and Code availability
Data from scRNAseq used in this study are from previously published datasets and are publicly available (see Key resource table). Scripts generated for this study are available upon request.

Mouse Strains
All mouse strains used have been previously described and included Crect, Krt14Cre, in the study, timed mating was set up, and the morning of the day a plug was detected was considered day 0. Genotyping was performed using standard protocols (See Supplemental Table   1 for specific primers and mouse strains). All experiments were approved by the NIH Animal Care and Use Committee.

Doxycycline treatment
Adult mice (6-8 weeks old, males and females) were fed Doxycycline diet (5001C w/6000ppm, Animal Specialties and Provision, PA, USA) ad libitum for 4 days (day 0), before changing the food back to standard diet. Tissues were harvested for analysis at indicated timepoints. For induction during embryonic development, females were fed doxycycline during pregnancy.

Organ Culture Explants
SMGs were dissected from ICR embryonic day 15 (E15) embryos and placed on Whatman well as a group of untreated glands. SMGs were cultured at 37°C in a humidified 5% CO2/95% air atmosphere for 24h (unless otherwise noted). RNA was isolated from homogenized glands before qPCR gene expression analysis as described below.
Organ culture experiments with washout and ligand treatments were cultured under the same conditions as described above. Here, glands were treated with UO126 (20µM) or vehicle (DMSO) for 24hrs before washout with fresh media 3x5 minutes. Glands were then incubated again for 24h with either UO126, DMSO, FGF7, and FGF10 (All recombinant FGF's from R&D Systems). Gentle but thorough mixing was done to assure adequate distribution of treatment in the media. Final dosages were 500ng for FGF7 and FGF10 (volume of 10µL).

Real time qPCR
RNA was isolated using either RNAqueous-4PCR total RNA isolation kit or RNAqueous-PCR micro kit with DNase treatment (Both from ThermoFisher). cDNA was made using the iScript cDNA Synthesis Kit (Bio-Rad) and 1 ng was amplified with 40 cycles of 95°C for 10 s and 62°C for 30 s. Gene expression was normalized to the house-keeping gene, Rps29. Amplification of a single product was confirmed by melt curve analysis and all reactions were run in duplicate.

Immunohistochemistry
For frozen sections, tissues or whole embryos were fixed in 2% paraformaldehyde Co-staining done with same species antibodies were performed using a multiplex method.
Paraffin sections were de-paraffinized as described above before antigen retrieval in a microwave Chloride, 0.2 mg/ml 4-Iodophenylboronic acid and 0.003% H2O2). Antigen retrieval step and staining steps were then repeated until all antibodies had been stained. Nuclear staining and cover slips were mounted as described above.
Hematoxylin and Eosin (H&E) staining of paraffine sections were performed by Histoserv Inc (Germantown, MD). Slides were scanned with a S60 NanoZoomer Digital (Hamamatsu) and images were exported using the NDP.view 2 software (Hamamatsu).

In situ Hybridization
Using

Computational analysis
Using previously annotated mouse SMG scRNAseq data, epithelial populations were computationally separated SEURAT's subset function. Epithelial subsets from E12 were renormalized and scaled to generate new SEURAT objects. E16 and P1 acinar subsets were integrated through standard pipeline to generate a new SEURAT object with mouse acinar cells.
All statistics for the computational analyses to determine significant markers were performed using the default pipeline statistical test in SEURAT. This analysis is based on non-parameteric Wilcoxon rank sum test and adjusted p-values of <0.05 were chosen as a measure of significance.
Human MSG datasets (GSE180544 and https://www.covid19cellatlas.org/) were integrated and previous annotation from the two datasets were used to identify cell populations. Human SMG and PG datasets were imported (GSE201333) and the previous annotations were used to identify populations.

Quantification of histological images
Images were taken from random areas of H&E-stained Krt5Cre; Fgfr1 fl/fl ; Fgfr2 fl/fl slides as described above. Duct/total area ratio was calculated by measuring the area of duct and total area using FJII [8]. For quantification of fluorescent images, imaging was performed using a 40x objective on a Nikon A1R+ MP microscope using resonant scanning method. From Krt5rtTA; tetCre;Fgfr1 fl ;Fgfr2 fl ;mTmG sections 10 random areas were selected (5µm thick confocal stacks with 0.5µm steps) from at least 3 different animals (n=3). The GFP and Hoechst expression intensity was measured by calculating the integrated density value of the samples histogram resulting from the maximum intensity projection. Quantification of the 10 points were averaged and GFP was normalized to Hoechst staining. Results were then averaged by biological replicates.
For quantification of Ki67, Cleaved Caspase3, SMGC and LPO, 2-3 confocal stacks (5µm stacks, 0.5 µm steps) from 3 glands (n=3) were processed using the denoise function of the Confocal NIS-Elements Package prior to quantification. Threshold with stack histogram was set manually to reduce background, and integrated density was measured on maximum projections and normalized to Hoechst (using FIJI). All quantification measurements of confocal imaging were performed blinded.

Statistical analysis
To compare more than two experimental groups, we performed one-way ANOVA with post hoc Dunnett's or Tukey test (as indicated) and for comparison of two data sets, the Student's t-test with two-tailed tests and unequal variance was used to calculate p-values. Graphs show mean ± SEM for each group from three or more replicates.      (B) Fgfr2b deletion in acinar cells leads to a decrease in gland weight ratio compared to wild type (Cre-). Graph shows mean ratio of gland weight over body weight with SD, n ≥3. One-way ANOVA with Dunnett's test for multiple comparisons to control (*p<0.05).
(C) Canonical acinar genes and genes enriched in Fgfr1b+ population were not affected by deletion of either Fgfr1b, Fgfr2b or both. Genes enriched in Fgfr2b+ cells were decreased after deletion of Fgfr2b or both receptors (n ≥3 for each genotype). One-way ANOVA with Dunnett's test for multiple comparisons to WT control (****p<0.001, ***p =0.0003,**p<0.003).
(E) Ablation of either Fgfr1b, Fgfr2b, or both did not affect localization pattern of SMGC (green), while LPO (red) was not detected after Fgfr2b deletion in P1 SMGs. Scale bars: 20µm.
(F) Deletion of either Fgfr1b, Fgfr2b, or both did not affect localization pattern of GSTT1 (green), while MUC10 (red) was not detected after Fgfr2b deletion in P1 SMGs. Scale bars: 20µm.  (C) Gene expression of canonical acinar markers were decreased after FGFR inhibition. Cldn10 and Bpifa2 were decreased after FGFR inhibitor. Aqp5, Bpifa2 and Lpo were also decreased after MAPK inhibitor. Genes enriched in the Fgfr1b+ population did not significantly change with the various inhibitors compared to vehicle control. One-way ANOVA with Dunnett's test for multiple comparisons to control (*p<0.05).
(D) Acinar differentiation was evident in all groups shown through IHC of AQP5, MIST1 and CLDN10. Scale bars: 50 µm.
(E) After 24hrs, SMGC could be detected in all groups, while LPO was not detected after FGFR and MAPK inhibitor treatment. Scale bars: 20 µm. (B) Genes reduced following MAPK inhibitor could be partially rescued by inhibitor washout.